Double-stranded ribonucleic acid as control against insects

ABSTRACT

A composition for use in formulations for controlling insect populations, including populations of mosquito and flies. The composition comprises one or more double-stranded constructs inhibitory to RNA transcription of ribosomal proteins. The invention also relates to method of using the compositions in formulations to inhibit insect populations.

BACKGROUND OF INVENTION

1. Field of Invention

The inventive subject matter relates to a composition and method for the control of mosquitoes and flies using double strand ribonucleic acid.

2. Background Art

Although RNAi has proven useful in functional studies for the knockdown and elucidation of individual gene effects in dipterans and has become a standard technique, development as a potential means of pest control has been slow.

Effective constructs have been fed directly to pests, expressed in bacteria, and expressed in plants (Zhu et al., 2011). Constructs have been developed and tested in Plutella xylosteia (Bautista et al., 2009) and Helicoverpa armigera (Mao et al., 2007; 2011) against upregulated transcripts that confer pesticide resistance to reintroduce susceptibility to traditional pesticides.

Larval life stages of insects appear to be generally more susceptible to RNAi (Huvenne & Smagghe, 2010), and RNAi-induced mortality in adult mosquitoes is difficult to reproduce although a few sporadic successes have been published (Isoe et al., 2011; Pridgeon et al., 2008). The difficulty of causing lethal phenotypes and the slow induction of critical deficits has caused others to target genes to induce susceptibility to pathogens (Campbell et al., 2008), make dipterans refractive to flavivirus infection (Aliyari et al., 2008), or produce short-term ovarian provisioning defects (Gulia-Nuss et al., 2011; Isoe et al., 2011; Sim & Denlinger, 2009).

Due to the importance of the ribosome in protein synthesis, transcripts encoding for these ribosomal proteins were examined using RNAi in several arthropod species with interesting results. In Drosophila melanogaster, knockdown of ribosomal protein S3a (RPS3a) resulted in inhibition of egg maturation (Reynaud et al., 1997) and knockdown of other ribosomal targets also resulted in inhibition of oviposition in adults or mortality in larvae. A novel study by Kurscheid et al (2009) in Rhipicephalus microplus individually knocking down three ribosomal transcripts as well as one proteasomal transcript resulted in a reduction in oviposition. Recently, this study was repeated in the Western predatory mite and also resulted in the reduction of egg laying but no change in mortality (Wu & Hoy, 2014). In a study of diapause induction, the knockdown of RPS3a in Culex species caused a short term reduction in the ability to provision eggs (Sim & Denlinger, 2010).

SUMMARY OF THE INVENTION

The current invention relates to one or more compositions, for inclusion into formulations for insect populations comprising double stranded RNA (dsRNA) constructs from aligned ribosomal transcripts. Examples of insects include mosquitoes, such as of Aedes aegypti and flies, such as Musca domestica.

In one embodiment, the dsRNA from mosquitoes comprises a 120-716 basepair (bp) construct encoding the ribosomal protein S6. In a preferred embodiment, the construct is 152 bp, but can be any length between 120 to 716 bp. Another embodiment comprises a 120-716 bp construct encoding the ribosomal protein L26, with a preferred embodiment of 162 bp. In one embodiment, the ribosomal sequences are produced by amplification using polymerase chain reaction (PCR) primers containing T7 promoter sequences, in order to enable production of dsRNA in available transcription systems. One or more of the compositions, comprising dsRNA encoding ribosomal proteins, can be included in a formulation for control of insects, such as for mosquito or fly populations.

The methods of delivery of these dsRNA molecules could be inclusion of the composition into formulations for application through spray equipment directly onto the insects. Alternatively, the compositions can be included in a natural or artificial carbohydrate containing bait or bloodmeal, by uptake from larval growth solution, or by application through spray equipment with various formulants. In one embodiment the compositions can operate over a range of concentrations, including from 5 ng/insect to 2000 ng/insect.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1: Effect of double-stranded RNA at specific inhibition of target gene expression. dsRPS6 and dsRPL26 result in specific knockdown in AAG-2 cells.

FIG. 2: Clutch size is reduced through multiple oviposition cycles in mosquitoes targeted with dsRNA against ribosomal transcripts.

FIG. 3: The effect of dsRPS6 and dsRPL26 on gene expression is long lasting.

FIG. 4: Dose curve of injected dsRPS6 shows significant differences in fecundity at 50 ng/org and above.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

Two major difficulties have limited practical development of RNAi-based pesticides for vector species; first, the necessity to develop a method to effectively deliver constructs in natural environments, and second, the development of constructs that consistently result in major alterations to the life history trajectory of the species, which could result in reduced disease. The inventive compositions address the prior art limitations by targeting transcripts of ribosomal protein S6 (RPS6) and ribosomal protein L26 (RPL26). The doubled-stranded RNA molecule, as used herein, is comprised of a sense strand, which is directly translated into protein, and a negative, complementary strand.

Example 1 Production of dsRNA

Three dsRNA constructs were manufactured and used for this study. Consensus transcript sequences for the small ribosomal subunit, RPS6, (AAEL000032), the large ribosomal subunit, RPL26, (AAEL005817) and as a control, Israeli Acute Paralysis Virus (dsIAPV) was used.

Constructs were manufactured following the instructions in the MEGASCRIPT® kit (Invitrogen™, Thermo Fischer Scientific Corporation, Waltham, Mass.) with a few modifications. Initial template was produced by PCR using Aedes aegypti Orlando female cDNA and T7-appended gene specific primers (Table 1). After gel purification of the initial product and sequencing to verify the expected amiplicon, this was used for further template amplification to produce sufficient quantity to allow input of 1 ug of template per 20 ul MEGASCRIPT® reaction. This amplified product was concentrated and buffer exchanged twice with nuclease free water in a spin concentrator (Amicon® 30K MW MWCO, Millipore®, Billerica, Mass.) to remove impurities left from the PCR reactions. The standard MEGASCRIPT® protocol was changed to allow the use of an entire kit of 20 reactions at one time using an input of 20 ug of the appropriate T7 template. The reaction was run for 24 hours at 37 C and then purified according to the protocol. Yields from this procedure using well were between 1.5-2.0 mg of each of the three constructs. Constructs were concentrated to 20 mg/ml using Amicon® 30K MWCO concentrators and then the concentrated dsRNA was frozen. This procedure was performed 3 separate times for dsRPS6 and dsRPL26 and twice for dsHP. A 480 bp dsRNA targeting a portion of the Israeli Acute Paralysis Virus (dsIAPV) (Maori et al., 2009), which is absent from mosquitoes was used as an additional control.

Samples of dsRNA for injection were diluted in MEGASCRIPT® elution buffer to a final concentration of 10 ug/ul with 0.003% Rhodamine B dye to visualize the injected solution. A preliminary series of injections with this concentration of dye was done to ensure that no toxicity or inhibition of oviposition would result.

TABLE 1 Primers for Aedes aegypti dsRNA construction and qPCR analysis Amplicon Name Sequence Size¹ SEQ No. T7F- taatacgactcactataggg 192 2 AAEL000032RB GTCCTGACCAACACCCGT T7R- taatacgactcactataggg 3 AAEL000032RB CCCTTCTTGACGACGATCAG T7F- taatacgactcactataggg 223 5 AAEL005817 CAAGAGCCGTAAGCGACATT T7R- taatacgactcactataggg 6 AAEL005817 ACCTGGACCACCTTACCGAC T7F- taatacgactcactataggg 253 220 AAEL009056 GTGTTTCGAATCCGTGTGAA T7R- taatacgactcactataggg 221 AAEL009056 ACCTGGCTTTGCATTCCTT qRpS6_F CTCGGCGAGTGTATGGAAAT 116 214 qRpS6_R CGTAGAAGTGACGCAGCTTG 215 qRpL26_F CCCTCTTTCCTTCCGACATC 105 216 qRpL26_R GGCGAGACGAGGAAACATT 217 qIAPV F GAGGCAGTAAAATTTCGCCA 218 qIAPV R AGGTGAAAGTCTTGCCATCG 113 219 ¹amplicon size of templates for dsRNA includes 40 bases contributed by the T7 promoters of the fusion primers that are not present in the final dsRNA product.

Example 2 Knockdown of Ribosomal Transcripts of Aedes aegypti

Analysis of the constructs in Table 1 was conducted using AAG-2 cells. AAG-2 cells were grown in Eagle's media (GIBCO) supplemented with 15% FBS (Lonza Inc., GA) in a 5% CO2 environment at 27 C. Cells were passaged weekly in T75 flasks with 10 ml of culture media. For experimental purposes, 200,000 cells were plated into each well of a 24-well flat-bottom tissue culture plate (3D, USA) after dissociation from the previous flask approximately 24 hours before use. Two replicate plates were made for collection and processing after 24 and 48 hours of exposure to dsRNA. Application of the two control dsRNA constructs as well as the two test constructs was made at 100 ug/ml in culture medium to 75-80% confluent AAG-2 cells. A previously characterized dsRNA targeting the inhibitor of apoptosis 1 transcript (dsIAP1) (AAEL009047) with known lethal activity against the AAG-2 cell line (Pridgeon et al., 2011; Liu & Clem, 2011) was used as a positive control at the same concentration. Samples were collected at 24 and 48 hours after application, washed with PBS (Gibco) and then frozen at −80 C or immediately used for RNA preparation.

In one embodiment, double-stranded RNA is utilized to knockdown ribosomal transcripts of insects, such as mosquitoes. In this embodiment, double-stranded RNA from 120 to 716 base pair region of ribosomal RNA from mosquitoes can be utilized. Although this example utilizes Aedes aegypti, any homologous ribosomal region, derived from other species, are contemplated.

In one embodiment, double-stranded RNA constructs are transcribed from nucleic acid sequences encoding ribosomal protein targets, RPS6 (AAEL000032) or RPL26 (AAEL005817), which are members of the small and large subunits, respectively. The sequences of the nucleic acid sequences whereby the double-stranded RNA are transcribed are given in SEQ ID No. 1 (RPS6) and SEQ ID No. 4 (RPL26). However, double-stranded RNA derived from other ribosomal proteins from other mosquito species can also be used. RNA can be transcribed from other nucleic acid sequences, from other mosquito ribosomal proteins, as in SEQ ID Nos. 7, 10, 13, 16, 19, 22, 25, 28, 31, 34, 37, 40, 43, 46, 49, 52, 55, 58, 61, 64, 67, 70, 73, 76, 79, 82, 85, 88, 91, 94, 97, 100, 103, 106, 109, 112, 115, 118, 121, 124, 127, 130, 133, 136, 139, 142, 146, 149, 152, 155, 158, 161, 164, 167, 170, 173, 176, 179, 182, 185, 188, 191, 194, 197, 200, 203, 206, 209, and 212.

In order to evaluate the effectiveness of the double-stranded RNA sequences at inhibiting transcription of ribosomal proteins expression of the large and small ribosomal RNA was quantitatively measured in the presence of the RNA. As a control, dsIAPV double-stranded RNA was synthesized using primers encoded by SEQ ID Nos. 220 (fwd) and 221 (rev). Quantitation of the expression of RPS 6, RPL26 and IAPV was evaluated using quantitative PCR using the primers encoded by SEQ ID Nos. 214-219 (Table 1).

We performed an initial analysis in the Ae. aegypti AAG-2 cell line to examine the specificity of our dsRNA triggers and ensure we were reducing transcript levels of the expected targets.

As illustrated in FIG. 1, AAG-2 cells treated with 100 g/ml of dsRNA trigger showed transcript specific reductions in gene expression, after 24 and 48 hours of exposure, cDNA prepared from treated cells were examined for relative expression of either RPS6 or RPL26. Results indicate that each dsRNA specifically reduced target expression but not the expression of the other two targets. As primer efficiencies ranged between 1.94 and 1.98, expression values were calculated using the ΔΔCt method of Livak & Schmittgen (2001). Amplification conditions and amplicons are as described in the methods. This data represents two replicate experiments. Error bars represent mean±SD.

We observed no change in morphology in the cells treated with the controls (dsGFP or dsMOSQ), dsRPS6 or dsRPL26 after 24 or 48 hours of exposure. We did see the expected morphological changes in the dsIAP1 treated cells (Pridgeon et al., 2011) within 24 hours and cell death by 48 h indicating that the system was functional. Samples were collected and analyzed for gene expression levels of RPS6, and RPL26 (FIG. 1). At 24 and 48 hours there was significant knockdown of RPS6 and RPL26 levels in cells treated with dsRPS6 or dsRPL26, respectively. No significant knockdown of RPS6 or RPL26 expression was observed in dsIAPV treated cells. We also did not observe significant knockdown of RPS6 expression in dsRPL26 treated cells or RPL26 knockdown in dsRPS6 treated cells indicating low crosstalk between the triggers. From this, we conclude the dsRPS6 and dsRPL26 triggers were accurately targeting the expected transcripts though we did not see visible morphological changes in the dsRPL26 or dsRPS6 treated cells.

Example 3 Evaluation of Mosquitoes Following Introduction of dsRNA

In these studies, glass capillaries were pulled to a fine tip using a KOPF® (Tujunga, Calif.) Model 720 needle puller with settings at 16.8 (resistance setting) and a pull strength of 4. A needle was placed into a NANOJECT™ 2000 (World Products Inc., FL) and the tip was broken to provide a sharp point. The device was set to deliver 100 On per injection. The needle was filled with 3-4 ul of a test solution containing either a control dsRNA (dsIAPV) or test dsRNA construct (dsRPS6 or dsRPL26). Three to five day old females were manually aspirated into a BIOQUIP® (Rancho Dominguez, Calif.) holding tube and cold anesthetized in a laboratory refrigerator at 3-4 C. Three replicate groups, of 11 mosquitoes each, were placed on the dorsal aspect on chilled slides placed into 15 cm petri dishes and aligned to allow easy injection. Plates and slides with aligned mosquitoes were maintained at 4 C until injected as we have not observed any mortality in this strain even after 24 hours of chilling. For injection, the slide with 11 mosquitoes was placed onto a BIOQUIP® Model 1614 chill table, the needle was primed and then introduced through the middle one-third of the mesokatepisternum. To visually ensure delivery of the solution, two measures were assessed during injection; the meniscus in the needle had to move upon injection and the pink color of the Rhodamine B must be visible through the cuticle at the ventral junction of the thorax and abdomen. If either of these measures were not seen, the mosquito was removed from the group. Successive injections followed the same procedure. Groups of approximately 10 mosquitoes were allowed to recover in an inverted screened cup (TK35™) (SOLO® (Dart Container Corporation, Road Mason, Mich.)) placed over sucrose saturated cotton. This recovery method, adapted from Isoe et al. (2011), resulted in less than 5% initial mortality in injected mosquitoes.

Oviposition Assays

An experimental timeline for these experiments is shown in Table 2. Cohorts of injected mosquitoes were allowed to recover for three days post-injection (PI) and then provided access to a warmed blood meal for 15 minutes or until all mosquitoes in the cup were visibly fed. Mosquitoes that did not feed were given an immediate second attempt with another warmed blood meal. If a mosquito did not feed after the second try, it was removed from the experiment. Blooded females were returned to 10% sucrose and maintained under standard temperature and humidity conditions. Two to three days post-blood meal (PBM) (6 d PI) the females were gently aspirated into individual oviposition cups (TK35™) (SOLO®) that contained 60 ml of deionized water and a 3 cm by 4 cm strip of soaked seed germination paper (Anchor Paper Co., Saint Paul, Minn.) for deposition of eggs. Oviposition cups were provided with 10% sucrose saturated cotton. Oviposition and mortality were tracked daily and eggs were counted after an additional 4 days at 10 d PI. Females were aspirated to new oviposition cups and given a second blood meal 2 days after transfer (12 d PI). Some individual mosquitoes from each group were collected and frozen for further analysis at 13 d PI. The same collection procedure was repeated after the second blood meal and eggs were counted at 20 d PI. Females were frozen for qPCR analysis after this second ovposition cycle in five of the six experiments.

TABLE 2 Experimental timeline and sample collection timepoints Day Post- Emergence Action  3 Cold anesthetize and inject dsRNA  6 Provide first bloodmeal  8 Split into individual oviposition cups 12 Collect eggs from first oviposition cycle 13 Collect organisms for qPCR 14 Provide second bloodmeal 20 Collect eggs from second cycle 21 Collect organisms for qPCR 23 Provide third blockmeal 29 Collect eggs from third cycle¹ Collect remaining organisms for qPCR¹ 30+ Rear embryonated eggs to examine viability ¹A third oviposition cycle was only tracked in the first of the six experiments. Due to increasing mortality in all samples after 20 days past-injection, this third cycle was abandoned.

In the first experiment, the mosquitoes were taken through the oviposition procedure for a third cycle rather than frozen. Some samples were collected at 29 d PI for analysis but the majority of mosquitoes in all groups had died so this was not repeated in future experiments.

Sample Preparation

Aedes aegypti (Orlando strain) were in continuous colony. They were collected near Orlando, Fla. in 1952. The Center for Medical, Agricultural and Veterinary Entomology CMAVE (Gainesville, Fla.) Aedes aegypti rearing protocol is highly standardized and has been described previously (Pridgeon et al., 2007; Clark et al., 2011). Initially, three one-hundredths of a milliliter of eggs are placed into 100 ml of deionized water along with about 50 mg of finely ground alfalfa/pig chow mixture. This container is placed under vacuum to deoxygenate for 2-4 hours and synchronize the hatch. The neonates are placed into a tray containing 3 liters of deionized water with 0.5 g of brewers yeast:liver powder (3:2). Trays are maintained at 27 C in an insectary under a 14:10 L:D cycle. Trays are fed 1 gram of 3:2 food on the following day and then again 2 days later. Pupae are collected into a 6 oz plastic cup and placed into a screened colony cage with 10% sucrose soaked cotton balls ad libitum. Under these conditions, early larval instars are completed daily, the 4^(th) instar requires 2 days, and pupal development takes two days. Mosquitoes emerge at the end of the seventh day after hatching and females weigh 2.5±0.3 mg (AVG±SD) each. Colonies were provided manually-defibrinated bovine blood as the protein source for colony maintenance. Mosquitoes were used for injection experiments 3-5 days after emergence and before taking a bloodmeal.

Mosquitoes for analysis were immediately frozen, transferred to microcentrifuge tubes and maintained frozen at −80° C. RNA isolation was performed using a room temperature column-based RNA isolation kit (Zymo Research Corporation (Irvine, Calif.)). RNA from individual mosquitoes was purified and subjected to a DNAse digestion following the manufacturer's instructions. RNA was eluted and checked for concentration on a NANODROP™ 2000 (Thermo Fisher Scientific (Waltham, Mass.)). The A₂₆₀/A₂₈₀ ratio was consistently greater than 1.95. RNA was stored at −80° C. To normalize the cDNA creation step, 300 ng of input RNA was used from each sample for first strand cDNA synthesis using the AMV-RT kit (Invitrogen™), Themo Fisher, Scientific (Waltham, Mass.) and oligo dT primers according to the manufacturer's instructions. After cDNA creation, samples were frozen at −20° C.

Quantitative PCR Analysis

Individual mosquitoes were collected after first, second, and third oviposition cycle and cDNA was prepared as above. Samples from each experiment were analyzed together on an Applied Biosystems® (Thermo Fisher Scientific (Waltham, Mass.)) STEPONE™PLUS™ using SYBR green chemistry and the standard recipe. Specifically, the 10 ul reaction was 5.0 ul of SYBR® Select (INVITROGEN™) (Thermo Fisher Scientific (Waltham, Mass.), 3.0 ul of combined 3 uM forward and reverse primers, 1.5 ul of nuclease free water, and 0.5 ul of sample cDNA. Primers for qPCR, (see Table 1, SEQ ID Nos. 214-219) were designed using the Primer3 server; the amplicon specified to be 85-125 bp, the T_(m) optimized for 60 C, and the amplicon was required to cross an exon/exon junction. The qPCR amplicon was also directed to a region outside that targeted by the dsRNA construct to prevent any possible amplification of residual T7 template. Reactions were run in at least technical duplicates in 10 ul volume. Melt curve analysis followed qPCR to ensure that only one product was being amplified. Although initial testing determined consistent results were produced when run under FAST conditions, standard cycling conditions were used. Each 96-well plate from a particular experiment was provided with a common control sample to allow plate to plate comparison. Relative quantification was performed by comparison to an L24 control gene which has been shown to be relatively consistent over the range of samples examined (Pridgeon et al., 2009; Choi et al., 2013). Comparison between samples used the 2^(−DDCt) method (Schmittgen & Livak, 2008) and test samples were normalized to expression of samples treated with elution buffer only. Template dilution curves were performed to ensure the primer efficiencies were adequately close to allow comparison using the Livak method.

Statistical Analysis

All statistical analysis were performed in JMP® (Statistical Analysis Institute (Cary, N.C.)) and methods were varied based on the character of the data and number of samples. To examine possible significant differences in oviposition levels of mosquitoes, percentages laying were sorted by dsRNA treatment group for both blood meals in each of the six experiments and then subjected to Kruskal-Wallis nonparametric analysis after initial data characterization indicated a non-normal distribution in some samples that produced low numbers of laying mosquitoes. Means separation was performed using Sennett's test.

Analysis of clutch size data showed normal distribution indicating the acceptability for use of a parametric analysis like analysis of variance (ANOVA). To identify significant interactions, pairwise comparisons were conducted between each category.

Effect of RPS6 and RPL26 dsRNA on Ovarian Morphology

Dissections of ovaries 36 hours after ingestion of a blood meal showed morphological differences based on the trigger injected. Ovarian dissection at 36 hours after a blood meal showed differences in egg provisioning. Provisioning of eggs was proceeding as expected for dsIAPV injected mosquitoes. By comparison, developing eggs in dsRPS6 injected and dsRPL26 injected organisms were delayed or reduced. DsRPS6 appeared to produce greater inhibition of egg maturation.

Representative ovaries from dsIAPV injected mosquitoes bad well developed follicles as would be expected at this stage of normal egg provisioning. The ovaries of dsRPS6 injected mosquitoes were reduced and immature although it appears that some follicles were slightly provisioned. Ovaries of dsRPL26 injected mosquitoes had some provisioning of some of the follicles but others were very immature and shrunken.

We next dissected and examined ovaries to determine if the effect observed at 36 hours PBM was also present after completion of the first oviposition cycle. The mosquitoes were 6 days PBM, at which time the controls had oviposited clutches of eggs on days 4 and 5 PBM. Dissection of ovaries again showed clear morphological differences. Remnants of the blood meal were not observed and had already been excreted from all treatments. Ovaries of control injected (dsIAPV or dsMOSQ) mosquitoes were empty or nearly empty after clutches of 48 and 63 eggs had been deposited. Dissection at six days after a blood meal (10 days post-injection), shows control dsRNA injected mosquitoes had empty or nearly empty ovaries and had laid clutches of normal size. Those injected with test dsRPS6 or dsRPL26 had laid no eggs and dissection showed only limited development in dsRPS6 treated and a mix of immature and more provisioned eggs in dsRPL26 treated.

In contrast, most mosquitoes targeted with ribosomal dsRNAs did not oviposit or laid a reduced clutch. Injection of the ribosomal dsRNA triggers resulted in immature and partially provisioned eggs. There was also a visible difference in ovary morphology between injections of dsRPL26 and dsRPS6 even though in both cases no eggs were laid. As observed at 36 hours PBM, the dsRPL26 treatment allowed more maturation than dsRPS6 treatment. Several nearly complete eggs were observed in the ovaries of the dsRPL26 treatment, however no eggs were laid during the normal oviposition period. These ovarian effects indicate that the provisioning defects induced by one introduction of these ribosomal triggers are able to persist through at least ten days.

Effects of dsRPS6 and dsRPL26 on Oviposition

We then sought to determine if dsRNA mediated knockdown of ribosomal transcripts in Ae. aegypti would have phenotypic effects like mortality (as in C. elegans) or reduced fecundity as in Rhipicephalus microplus (Kurscheid et al., 2009) and Metaseiulus occidenttalis (Wu & Hoy, 2014). After dsRNA injection and subsequent blood feeding, we detected no differences in mortality between treated and control groups through 20 days of observation with less than 20% mortality in all cohorts. Mortality in all cohorts increased dramatically after day 20 but was not different between treatments. The normal laboratory lifespan of the Orlando strain is about 30 days. We did observe significant reductions in fecundity in those cohorts treated with dsRPS6 and dsRPL26 through two oviposition cycles.

Effects on Clutch Size

We also examined whether the treatments with ribosomal specific triggers had an effect on clutch size through multiple oviposition cycles. Eggs laid by all mosquitoes from the six experiments were grouped by trigger and clutch size was plotted (FIG. 2). Eggs were counted for each organism after each oviposition cycle. Clutch size was significantly reduced after the first (A) and second (B) oviposition cycles. After the second cycle, many of the mosquitoes treated with dsRPL26 or dsRPS6 still produced no eggs or only a few eggs. Results of a third oviposition cycle (C) are shown but sample sizes were too small for statistical analysis. With the exception of (C), the data here represents combined data from six independent experiments. Significant differences are denoted by different letters over the columns. Error bars represent mean±SD.

In FIG. 2, Average clutch sizes varied significantly between the controls and test (dsRPL26 and dsRPS6) triggers (K-W test, P=<0.001, H=184.372, df=3). Average clutch sizes were calculated from the combined output of six independent experiments for the first and second oviposition cycles. An average of 54.9±33.1 (AVG±SD) eggs were produced by the dsIAPV treated and 53.3±35.7 eggs were produced by the dsMOSQ controls. The average clutches for the test dsRNA treated mosquitoes were significantly smaller at 1.21±2.3 for dsRPS6 and 4.3±10.4 for dsRPL26 treated mosquitoes after the first cycle (FIG. 2A). The two ribosomal dsRNAs were not significantly different from one another in pair-wise comparison. Inhibition of the total reproductive output based on these clutches was considerable; the roughly 160 mosquitoes in the control dsIAPV and dsMOSQ groups laid 8170 eggs, the 187 mosquitoes injected with the test constructs, dsRPL26 and dsRPS6 laid 471 eggs through the first cycle for a 94.2% reduction in total output. Individual samples were collected from each group in various experiments for later gene expression analysis.

Samples sizes for the second oviposition cycle are smaller based on natural death of mosquitoes, removal of those that did not take a second blood meal, and removal of live mosquitoes for dissection, RNA preparation, and gene expression analysis after the first cycle. In this case, the combined total number of mosquitoes for each construct ranged from 46-64. The results of the second cycle of blood feeding and oviposition showed a still significant (K-W test, P=<0.001, H=56.65, df=3) although a weaker effect (FIG. 2B). Control treatments (dsIAPV and dsMOSQ) both had similar mean clutch sizes of 63.5±30.1 and 66.3±31.0 eggs respectively. This was significantly different from the clutch size of dsRP S6 (22.4±32.6) and dsRP L26 (25.7±33.3) injected cohorts, which as before, were not significantly different from one another. It is interesting to note but not unexpected that the clutch size was consistently variable among the four groups and was also similar to the variability in clutch size seen in untreated Aedes aegypti indicating a large natural variation in fecundity. Total effect on output was again large but not of the same magnitude as observed during the first cycle. Although the sample size of the mosquitoes injected with dsRPS6 and dsRPL26 (n=96) was slightly larger than the 92 surviving control injected mosquitoes, the overall reproductive output was 2430 versus 5959, or still more than a 40% reduction.

Considering only the mosquitoes that survived through this second gonotrophic cycle, the overall effect on egg production through two cycles was impressive. The 92 control mosquitoes had a combined output from both cycles of 10848 eggs versus 2734 laid by both test groups (RPS6 and RPL26) for an overall 74.8% reduction over the life of the mosquitoes. The overall average fecundity was significantly different between the controls and test constructs (ANOVA, P<0.0001, F=72.08, df=3,197). Double stranded-RNA IAPV treated mosquitoes averaged 119.0±50.2 eggs and dsMOSQ treated averaged 116.8±57.2 for both cycles combined. Those injected with the ribosomal protein targeting triggers dsRPS6 and dsRPL26 averaged 20.8±33.6 and 31.1±37.7 eggs respectively over the two cycles. Eggs collected from both cycles of this first experiment were allowed to embryonate and then tested for viability. We observed no significant differences in the percentage of either hatching or adult emergence between eggs laid by controls or test injected cohorts.

Gene Expression in Ae. aegypti Orlando Strain

We examined gene expression levels of the two targeted ribosomal transcripts after treatment with dsRNA. As no effect on fecundity was observed in siRPS6 treatments, we did not include these samples in the gene expression analysis. Samples collected after the first blood meal showed a significant reduction in expression levels as compared to control dsRNA injected samples (FIG. 3). In FIG. 3, quantitative PCR analysis of samples collected after first, second and third oviposition cycles were examined for expression levels of A) RPL26 and B) RPS6. Significantly reduced expression in dsRNA-treated samples was evident after each blood meal showing a lengthy effect from a single injection of dsRNA. Calculation of RE was performed using the 2̂ddCT method with the transcript level of L24 as the control gene. Efficiencies of test and control gene primer sets are from 1.94 to 1.98. Different letters represent significant differences (P<0.05) within each group. Error bars represent means±SD.

The dsIAPV and dsMOSQ injections did not result in knockdown of either the RPS6 or RPL26 transcripts. For those cohorts treated with the dsRPS6 trigger, significant knockdown was observed at both 13 and 20 days PI (FIG. 3A). Significant RPL26 knockdown was observed at d13 (FIG. 3B). By d20, some knockdown was observed but was not significant when compared to dsIAPV. However, it was significant when compared to the other control, dsMOSQ.

Effect of dsRPS6 Doses on Fecundity

We performed the same oviposition assay as described above over a range of lower doses of dsRPS6 than the initially tested screening dose of 1000 ng to examine the effects on oviposition (FIG. 4). In FIG. 4, injection of doses of dsRPS6 from 1000 ng/org to 50 ng/org resulted in significant reductions in fecundity in comparison to dsGFP injected cohorts (1000, 100, and 50 ng/org). Fecundity was not different than control injections at 25 ng/org of injected dsRPS6. A bloodmeal was provided three days after injection and eggs were counted on 10 days post injection. Error bars represent mean±SD from 36-81 individual organisms per dose. Columns with asterisks represent significant differences (P<0.05) from the 1000 ng/org dsGFP injected controls.

We found that injection of doses as low as 50 ng resulted in significant reductions in egg production when compared to dsGFP injected cohorts (KW test H=176.95, P<0.001, df=9). A significant difference was not observed at a dose of 25 ng. Three different doses of dsGFP also did not differ significantly. Interestingly, the mean clutch size was no different between the initial screening dose of 1000 ng and doses of 400 ng and 200 ng with an average of five eggs. At doses below 200 ng, the average clutch size began to increase.

We determined that dsRNA triggers directed against representative transcripts involved in the small or large ribosomal subunit (RPS6 or RPL26) were effective and sufficient to cause nearly complete shutdown of oviposition after blood feeding. This decrease in egg laying behavior correlates well with levels of gene expression measured from whole body preparations, and there appears to be no obvious fitness effect from knockdown of these specific ribosomal transcripts until ingestion of a blood meal. After ingestion, the organism exhibits no additional mortality even though suffering severe effects on fecundity. We also show that an siRNA trigger directed against RPS6 does not have the same effect even though it was delivered in a mass equivalent and by the same method indicating some possible selectivity in uptake. Additionally, we show one injection is long lasting (greater than 20 days), causing effects weeks afterward though a reduction of the effect occurs over time.

In contrast to other results, which also noted mortality along with fecundity decreases, no effect on general fitness was observed during these ribosomal targeting experiments. The administration of either dsRPS6 or dsRPL26 did not cause increased mortality in the adults compared to the controls. They exhibited normal behaviors; they flew, bloodfed, and excreted blood meal products even though examination by qPCR clearly showed that the specific gene targets were being affected. Even three weeks after injection, no increased mortality was observed as might be expected in an organism unable to produce the targeted ribosomes or only able to produce transcript at a reduced rate. A recent thorough study in mouse cell lines indicated that most expression regulation (>73%) is at the transcript level even though protein half-life and transcript degradation play small roles in overall phenotypic effect of gene knockdown (Jovanovic et al., 2015). It is likely the regulation effects may not be the in the same proportions in insects as it is in mammals but there is little work examining these issues in insects. Even in the quickly reproducing AAG-2 cell line, gene knockdown did not cause an increase in cell mortality as would seem to be appropriate in a tissue that was continually required to make new proteins to provision daughter cells.

A phenotypic effect that presents only after resources have been provisioned for reproduction has been observed in at least two organisms. In M. occidentalis, no mortality was noted and no gene knockdown was observed after sugar-feeding the targeted dsRNA. However, gene knockdown was observed after the return to prey, required by this obligate predator for reproduction (Wu & Hoy, 2014) and subsequent fecundity was greatly reduced. In the mosquito, knockdown of coatomer proteins caused no mortality until the taking of a blood meal (Isoe et al., 2011). The mechanism at work behind this delayed effect is unclear. It is possible that sufficient quantities of proteins are present for day to day maintenance so transcript requirement for new synthesis would generally be low. However, taking of nutrition adequate for reproduction would require the synthesis of additional transcript to make additional proteins for provisioning of eggs. For the mite, it is proposed that an inhibitory effect results from the high sugar concentration in which the dsRNA was administered resulting in the lack of observed transcript knockdown. Our triggers were delivered by injection and therefore sucrose mediated inhibition is unlikely. Expression analysis after a blood meal of the ribosomal transcripts does not show a large spike in these levels post-blood meal as would be expected if new ribosomal transcript synthesis began (Dissanayake et al., 2010) We confirmed this with several screens of post blood-fed time point series (data not shown). An interesting possibility is that mRNAs for the ribosomal transcripts are bound in the fat body by ribonucleoprotein particles (Niu & Fallon, 2000) which may make them inaccessible to RNA induced silencing complex (RISC) cleavage. However, at this point, the mechanism behind the effect we have observed is unclear and requires further study.

We did not observe the same biological effect of reduced fecundity when siRNA targeting RPS6 was injected although dsRNA against the same target (RPS6 or RPL26) produced long-lasting effects. Both trigger formats were injected into the hemolymph but had different effects. This may indicate that the mechanism to take exogenous dsRNA into the cell from the hemolymph may have a lower size limit. Bolognesi et al. (2012) noted that dsRNA size was a critical factor in dsRNA efficacy in the western corn rootworm. It has been noted that uptake of siRNA from the gut of some insects is not as effective as dsRNA triggers (Whyard, 2015; Bolognesi et al., 2012). Intriguingly, this effect in insects is the opposite of that observed in mammals where siRNA are effective while longer dsRNA activate the interferon system leading to a general shutdown in gene expression as an antiviral response mechanism.

The use of RNAi triggering molecules as therapeutic or control agents has been advancing in mammals and plant feeding insects. In species like mosquitoes that vector human or livestock diseases, progress has been much slower. The best triggers would cause quick mortality but these have been difficult to reliably identify. Although, in the current study, significant reductions in fecundity are not as immediately effective as a traditional chemical pest control method against a vector species, the massive reductions in egg output coupled with the natural sources of mosquito mortality could quickly reduce a vector population if the construct could be effectively delivered. Future work will investigate the methods to deliver these constructs through feeding (Zhang et al., 2010; Coy et al., 2012; Whyard et al., 2015) as well as work to understand specific tissue-level impacts behind the effect observed.

Example 4 Validation of RPS6 and RPL26 Targeting in the Housefly

To assess the validity of targeting species specific versions of ribosomal protein transcripts as a control method of another species, we produced a M. domestica specific constructs targeting RPS6 (SEQ ID No. 230) and RPL26 (SEQ ID No. 231). The constructs were produced in the manner described above using the primers listed in Table 3. The double-strand RNA constructs were made as for mosquitoes in Example 1, using the primers SEQ ID Nos. 222-225. Quantitation of RPS6 (SEQ ID No. 226/227) and RP26 (SEQ ID No. 228/229) was by quantitative PCR using qMdom RPS6 or qMdom RPS26 primers as in Table 3. Constructs were delivered by microinjection in an initial test dose of 5 ug/female fly. Recovered flies were maintained on sucrose for three days before being given access to a protein containing food sufficient for oviposition. We clearly observed the same effect on oviposition seen in mosquitoes. Dissection of gravid flies showed significant defects in ovarian provisioning in the flies that had been treated with the fly specific dsRPS6 (Mdom dsRPS6) relative to the controls. Injection of 5 ug/org of dsRPS6 resulted in significant reductions in fecundity in comparison to control organisms. Cohorts of female M. domestica were either not treated, injected with nuclease free water, injected with dsGFP, or injected with the described fly specific version of dsRPS6. After three days recovery, a protein source was provided to allow provisioning of eggs, ovaries were dissected and photographed. Note that the ovaries from dsRPS6 treated M. domestica have little to no ovarian development while controls are fully provisioned.

TABLE 3 Primers for Musca domestica dsRNA construction and qPCR analysis Amplicon SEQ ID Name Sequence Size¹ No. T7F-MdomRPS6 taatacgactcactataggg 202 222 TGAAACAGGGTGTCCTTAGC T7R-MdomRPS6 taatacgactcactataggg 223 CCCTTCTTGACGATGACCAA T7F-MdomRPL26 taatacgactcactataggg 222 224 AAAGAACCGCAAGCGCCATT T7R-MdomRPL26 taatacgactcactataggg 225 GCTTGGACAACCTTGCCAAC qMdomRPS6_F GGTGTTCCTCCAAATCAGACA 131 226 qMdomRPS6_R ACTTGGCCCATACGCTTCTC 227 qMdomRPL26_F ATGCCAACGGTACCAACGTT  86 228 qMdomRPL26_R AGCTTTGCGATCCTTGTCCA 229 ¹amplicon size of templates for dsRNA includes 40 bases contributed by the T7 promoters of the fusion primers that are not present in the final dsRNA product.

Defects were not observed in those treated with control dsRNA (dsGFP) or injected with only injection buffer when compared to the ovaries of untreated flies. As in Ae. aegypti, this defective ovarian provisioning resulted in significant reductions in fecundity. Untreated flies produced an average of 122 eggs/female (N=51) which was similar to that of dsGFP treated cohort (136.96 eggs/female, N=53). Flies treated with M. domestica-specific dsRPS6 produced less than 10% of the eggs of the controls (8.14 eggs/female, N=28). This proof of concept shows that targeting RPS6 transcripts is effective in species of insect.

Example 5 Method for Control Mosquito or Fly Proteins

It is contemplated that the dsRNA can be used in a method to control insect populations, for example mosquito or fly populations. The method comprises exposing insect populations, such as breeding mosquito or fly populations, to formulations containing one or more dsRNA constructs targeting small or large ribosomal protein transcripts. The populations can be exposed to dsRNA to either the large, small or both large and small ribosomal proteins.

In order to expose the insect populations, dsRNA to insect ribosomal proteins can be included in insect bait for oral delivery of dsRNA. Alternatively, dsRNA can be incorporated into suitable insect expression systems, such as baculovirus expression systems. The expression system, incorporating nucleic acid sequences to either the small or large ribosomal proteins can then be included into insect bait or provided and exposed to insect populations via a spray or other application method.

As an example, dsRNA to the large and/or small ribosomal subunits, produced using the primers as in Table 1 or Table 3, are envisioned. Alternatively, the formulation can comprise one or more dsRNA molecules capable of inhibiting any of the ribosomal proteins. Exposure of the mosquito or fly populations to dsRNA inhibits ribosomal transcription.

The inventive method provides inhibition of oviposition in the populations, thereby reducing future populations. Clutch size is reduced through multiple oviposition cycles after an initial exposure. Maintenance of low mosquito or fly populations can be obtained by regularly treating areas with preparations containing dsRNA.

In the inventive method, the composition comprises one or more double stranded RNA molecules. The dsRNA molecules are transcribed from PCR produced ribosomal nucleic acid molecules encoding any of the ribosomal proteins. For example, the double stranded RNA molecules can be transcribed from the nucleic acid sequences selected from the group consisting of SEQ ID Nos. 1, 4, 7, 10, 13, 16, 19, 22, 25, 28, 31, 34, 37, 40, 43, 46, 49, 52, 55, 58, 61, 64, 67, 70, 73, 76, 79, 82, 85, 88, 91, 94, 97, 100, 103, 106, 109, 112, 115, 121, 124, 127, 130, 133, 136, 139, 142, 146, 149, 152, 155, 158, 161, 164, 167, 170, 173, 176, 179, 182, 185, 188, 191, 194, 197, 200, 203, 206, 209, 212 for mosquitoes or SEQ ID Nos. 230 and 231 for house flies.

In one embodiment, dsRNA encoding the small ribosomal protein, can be used to control mosquito populations, wherein the dsRNA is produced by transcription of the nucleic acid sequence of SEQ ID No. 1, produced by using the forward polymerase chain reaction primer SEQ ID No. 2 and the reverse polymerase chain reaction primer SEQ ID No. 3. Similarly, the large ribosomal protein can be used, wherein it is transcribed from the nucleic acid sequence of SEQ ID No. 4, produced using the forward polymerase chain reaction primer SEQ ID No. 5 and the reverse polymerase chain reaction primer SEQ ID No. 6. Production of dsRNA can be made by any method for making double stranded RNA. These compositions, alone, or together, can be incorporated into anti-insect formulations designed to control the insect populations, for example mosquito or flies.

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What is claimed is:
 1. A composition for controlling insects, comprising one or more double-stranded RNA molecules, wherein said double-stranded RNA molecules are transcribed from nucleic acid sequences encoding insect ribosomal proteins.
 2. The composition of claim 1, wherein said insects comprise mosquitoes and flies.
 3. The composition of claim 1, wherein said composition comprises one or more double-stranded RNA molecules, wherein said double-stranded RNA molecules are transcribed from nucleic acid sequences selected from the group consisting of SEQ ID Nos. 1, 4, 7, 10, 13, 16, 19, 22, 25, 28, 31, 34, 37, 40, 43, 46, 49, 52, 55, 58, 61, 64, 67, 70, 73, 76, 79, 82, 85, 88, 91, 94, 97, 100, 103, 106, 109, 112, 115, 121, 124, 127, 130, 133, 136, 139, 142, 146, 149, 152, 155, 158, 161, 164, 167, 170, 173, 176, 179, 182, 185, 188, 191, 194, 197, 200, 203, 206, 209, 212, 230, and
 231. 4. The composition of claim 1, wherein the ribosomal protein is the small ribosomal protein
 5. The composition of claim 1, wherein said ribosomal protein is the large ribosomal protein.
 6. The composition of claim 1, wherein said double-stranded RNA is incorporated into insect bait.
 7. The composition of claim 1, wherein said double-stranded RNA molecule is included in and expressed from a viral vector.
 8. The composition of claim 7, wherein said viral vector is baculovirus vector system.
 9. A method of controlling insect populations, comprising inhibiting RNA encoding ribosomal proteins by exposing insects to a formulation comprising the composition of claim
 1. 10. The method of claim 9, wherein said formulation containing the composition of claim 1 is incorporated in insect bait.
 11. The method of claim 9, wherein said composition of claim 1 is expressed from an insect vector
 12. The method of claim 11, wherein said insect vector is baculovirus expression system.
 13. The method of claim 9, wherein said formulation comprises one or more double-stranded RNA, wherein said double-stranded RNA molecules are transcribed from the nucleic acid sequences selected from the group consisting of SEQ ID Nos. 1, 4, 7, 10, 13, 16, 19, 22, 25, 28, 31, 34, 37, 40, 43, 46, 49, 52, 55, 58, 61, 64, 67, 70, 73, 76, 79, 82, 85, 88, 91, 94, 97, 100, 103, 106, 109, 112, 115, 121, 124, 127, 130, 133, 136, 139, 142, 146, 149, 152, 155, 158, 161, 164, 167, 170, 173, 176, 179, 182, 185, 188, 191, 194, 197, 200, 203, 206, 209, 212, 230, and
 231. 14. The method of claim 9, wherein said double stranded RNA target transcripts encoding ribosomal proteins and include the small ribosomal protein and the large ribosomal protein. 